Chemotherapeutic agents, such as the epipodophyllotoxins and the anthraeyclines, target DNA topoisomerase II (topo II), and inhibit the re-ligation function by trapping topo II in a DNA-cleavable complex. Approximately 8% of cancer patients who are treated with topo II inhibitors develop therapy-related acute myeloid leukemia (t-AML), or myelodysplastic syndrome (t-MDS), many of which involve the MLL gene at 1lq23 or the RUNX1/AML1 gene at 21q22. We have already shown that the breakpoints in MLL, AF9 (the most common MLL partner), AML1 and ETO (the most common AML1 partner) tend to cluster in breakpoint cluster regions (BCR). We will map and clone the genomic breakpoints in two other partner genes, namely CBP and MDS1/EVI1 involved with MLL and AML1, respectively, in t-AML patients. We have proposed that there is a common mechanism for chromosome translocations mediated by non-homologous recombination occurring in "hot spot" regions, in both de novo and therapy-related leukemia. Our objective is to investigate whether there are common structural elements in the BCRs of MLL and AML1 and their most common partner genes, as well as those partner genes that are involved almost exclusively in tAML or t-MDS. We have determined the location of particular structural elements in theMLL BCR and in AF9 at 9p22 as well as inAML1, and the ETO gene at 8q22. We propose to begin similar analyses of the CBP gene at 16p13 and the MDS1/EVI1 gene at 3q26. The structural elements of interest for these six genes include: (1) in vivo topo II DNA cleavage sites induced after treatment with chemotherapeutic drugs; (2) DNase I hypersensitive sites (HS); (3) scaffold associated regions (SARs); (4) S1 and Mung bean single stranded DNA regions; and, (5) the precise sequence of the DNA cleavage sites oftopo II, S1, Mung bean, and MNase. Our specific aims include the completion of localization of these elements in AF9, AML1, ETO, CBP and MDS1/EVI1, and the determination of the structural similarities between the BCRs in these five genes and in MLL. We will also correlate the location of these structural elements with BCRs in all six genes. Our research should lead to a better understanding of the mechanisms giving rise to the genetic rearrangements involved in the pathogenesis of therapy-related leukemia, and may have ramifications for the etiology of other recurring translocations in human tumors.